EA023046B1 - USE OF HEAT KILLED MYCOBACTERIUM w FOR REDUCING INDUCED TLR ACTIVITY - Google Patents

Abstract

Mycobacterium w or its components are found to have poly-TLR-antagonistic activity to induced TLRs (toll-like receptors) by varieties of TLR ligands. The induced TLR against which inhibitory effect is seen includes TLR 3, 4, 5, 6, 7, 8, 9. They also display antagonistic activities to effects of TLR ligands. They are also useful in management of diseases wherein TLRs are overexpressed, like sepsis, multiple sclerosis, optic neuritis, chronic obstructive pulmonary disease, multiple myeloma etc.

Description

According to the present invention, there is provided a means of reducing the induced TK activity by using MyoBactyl ν (mycobacteria ν) or its components, and treating diseases associated with TK antagonists by using the mycobacterium ν or its components.

BACKGROUND OF THE INVENTION

The present invention provides an antagonist of a poly-U11-like receptor (TB). To11-like receptors (TB) are a family of proteins. The innate immune system recognizes pathogens and initiates an effective and appropriate response through TB. TBs are part of the large 1L-1K / TB-superfamily, which includes 1L-1K (interleukin 1 receptors), 1L-18K and a group of orphan receptors. The family is determined by the presence of the cytoplasmic domain of the To11-like / 1L-1 receptor responsible for resistance (To11-bke-1L-1 hexMaps (T1K) iotash), which is responsible for mediating signal transmission in the forward direction. To date, 13 TB have been identified; TLC 1-9 are common to mice and humans, while TK.10 functions only in humans, and TK 11, 12, and 13 are found only in mice. For many, but not all of these receptors, a role has been established in the initial detection of specific pathogen-associated molecules (RAM) and the response to these RAMs.

In macrophages and neutrophils, this stimulates innate immune responses, such as inflammation and the induction of bactericidal activity, while activation of TB expressed on dendritic cells triggers adaptive immunity by inducing 1L-12 and costimulatory molecules.

Despite the fact that TLCs are part of the body's defense system, their increased expression is associated with a number of diseases. One such disease is sepsis. It is estimated that in the United States, the occurrence of sepsis due to infection is approximately 750,000 cases per year with an average mortality rate of approximately 30% (Apdic e! A1. . Endotoxin or lipopolysaccharide (Lp§), which is the main component of the cell wall of gram-negative bacteria, is an agent that causes gram-negative sepsis. Endotoxin induces an innate immune response mainly through the U11-like receptor 4 (TK4) (Mei / 1Show e! A1., NaYuGe Uo. 388 (6640), (1997), pp. 394-397) in infected hosts, whereby the body receives a warning about a bacterial infection, thus causing an antimicrobial attack by the host's immune system. Typically, such an immune response has a healing effect on infected hosts, but an excessive immune response to endotoxin can be pathological, leading to a systemic inflammatory response syndrome (8HC§), organ failure, severe sepsis, and possibly septic shock and death. Symptoms of these conditions include fever, diffuse inflammation, and more severe conditions, such as disseminated intravascular coagulation (I1C), hypotension, acute renal failure, acute respiratory distress syndrome (AKP§), hepatocellular damage, and heart failure.

To11-like receptors expressed under the action of Lp§, in addition to TK.4, include other TK 2, 9. Since endotoxin itself is a highly heterogeneous molecule, the manifestation of many toxic properties of endotoxin is explained by the presence of a highly conserved hydrophobic part of lipid A. An effective drug that acts in as an antagonist of TK4 and which is an antagonist to this conservative structure of lipid A, is known as E5564 (also known as compound 1287, § CEA and erytoran) (Mi11agkeu e! a1., 1. Pba tasok Exp T1eg 304 (3):.. 1093-1102, 2003). This drug is described as Compound 1 in US Pat. No. 5,681,824, ΧΘ / 2004/071465 (Mekoik apy kk k ogika ίη Not ад ок п ап ап к ак ак еп х х еп еп иа иа (Methods and kits for use in the diagnosis and treatment of endotoxemia)) , which describes a method for determining whether a patient can benefit or continue to benefit from treatment with an antagonist of the U11-like receptor 4 (TK4). It is also known that inhibition of the To11-like receptor 4 by erythoran weakens myocardial ischemia-reperfusion injury. C1gci1aiop 114 (1 §irr1., (2006), p. 1270-4.

Other diseases that are characterized by increased expression of one or more TBTs include the following:

1) exacerbation of latent or active viral infections (e.g. HIV infection, cytomegalovirus, herpes simplex virus and influenza virus),

2) a congenital or acquired predisposition to pulmonary bacterial infection,

3) congestive heart failure with pulmonary edema,

4) chronic obstructive pulmonary disease,

5) multiple myeloma,

6) §ЬЕ (systemic lupus erythematosus),

7) lupus erythematosus,

8) ulcerative colitis,

9) Crohn's disease,

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10) autoimmune diseases,

11) rheumatoid diseases

12) chronic hepatitis,

13) malaria (R. GaShraglt),

14) multiple (multiple) sclerosis,

15) retrobulbar neuritis (optic neuritis),

16) viral encephalitis (West Nile),

17) candidiasis,

18) atherosclerosis, but this is not limited to.

Some of these disorders respond to conventional therapy, while for most diseases, final therapy does not exist.

Selected antibacterial, anti-inflammatory, and immunomodulating adjuvant therapies were studied in patients with severe sepsis and septic shock.

Type of therapy Neutralization of microbial toxins

Target (s)

Endotoxin

Agents Antibodies against endotoxin, antibodies against lipid A, lipopolysaccharide analogues, agents for excretion of lipopolysaccharide

Nonspecific Numerous High corticosteroids anti-inflammatory and inflammatory and dose, corticosteroids in immunomodulatory immune mediators low dose medicines pentoxifylline. immunoglobulins interferon gamma Inhibition Pro-inflammatory Antibodies Against Factor specific cytokines: factor tumor necrosis, mediators tumor necrosis soluble receptors tumor necrosis factor Interleukin-1 Receptor antagonist interleukin-1 Phospholipid Phospholipase inhibitor Components: A2 Phospholipase A2 Cyclooxygenase Ibuprofen T romboxane Dazoxiben, Ketoconazole Activation factor Factor antagonists platelet count platelet activation acetylglycosylase factor platelet activation Free radicals L / Acetylcysteine, Selenium oxygen Nitrogen oxide L / -methyl-L-arginine Bradykinin Bradykinin antagonist Adjustment Antithrombin III, Activated Protein C coagulopathy path inhibitor coagulation cascade tissue factor

Despite all of the above, the incidence associated with sepsis does not decrease. Thus, there is a need to offer better therapeutic options for such diseases.

Mycobacteria \ ν is a non-pathogenic, cultivated, atypical mycobacterium with biochemical properties and rapid growth characteristics similar to those of group IV mycobacteria according to the Kipuop (Runyon) classification.

It has been found that it has common antigens with Musoacantherope and Musoacantherocereus. It was found that with its help, leprosy is prevented in humans by converting individuals with a negative reaction to lepromine into individuals with a positive reaction to lepromine. It was also found that with its help the prevention of tuberculosis in animals is provided. In addition, it was found that with leprosy its use leads to a decrease in the duration of therapy in relation to bacterial killing, clearance, as well as to a decrease in the duration of the course of treatment in the clinic when used in conjunction with multidrug therapy. A pharmaceutical composition containing mycobacterium ν has been approved for human use since 1998 in India.

All of this is described in various patents and publications. Heat Inactivated Mycobacterium ν is available in India. It contains 0.5 x ^{10 9} cells of heat-inactivated mycobacteria ν per 0.1 ml of the pharmaceutical composition.

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Object of the invention

The objective of this invention is to reduce the induced TK activity by using mycobacteria \ ν or its components.

Another object of the invention is to provide the poly-TK antagonistic activity of mycobacteria or its components upon induction by known synthetic TK agonists, such as CpC, ΘΌΝ (oligodeoxynucleotide), or natural, such as lipopolysaccharide.

Another objective of the invention is to provide the antagonistic activity of mycobacteria ν and its components in relation to the action of TK ligands, such as lipopolysaccharide, E-e and

etc.

Another objective of the invention is to ensure the usefulness of mycobacteria ν or its components in the treatment of diseases in which there is an increased expression of TB.

Another object of the invention is to provide mycobacteria ν or its components for the treatment of a disease like sepsis, multiple myeloma, malaria, multiple sclerosis, retrobulbar neuritis, chronic obstructive pulmonary disease, in order to improve the morbidity and mortality associated with them.

A brief description of the graphic materials

FIG. 1. The effect of mycobacteria ν on the induction of TB through the action of L8.

FIG. 2. The effect of mycobacteria ν on the poly-TK ligand upon induction of TK.

FIG. 3. The effect of mycobacteria ν on the action of the Ligand L8 in induced hyperthermia (pyrogenicity test).

FIG. 4. The effect of a composition containing mycobacterium ν on sepsis induced by E. soy (intraperitoneal administration + high dose antibiotic).

FIG. 5. The effect of a composition containing mycobacterium ν on sepsis induced by E. soy (intraperitoneal administration + low dose antibiotic).

FIG. 6. The effect of a composition containing mycobacterium ν on sepsis induced by E. soy (intravenous administration).

Detailed description of the invention

It is known that pharmaceutical compositions containing mycobacterium ν and / or its components provide a Ty1 response. It is also known that they have common antigens with MusoBaSetst 1ergae and MusoBaShetst 1iEegsiO818. It was found that they are useful in the treatment of leprosy, improving the killing of microorganisms and their clearance from the body, which leads to a faster recovery. It was also found that they are useful as a prophylactic against tuberculosis and leprosy.

It was unexpectedly discovered that they also have unique properties to reduce the activity of TB. Their effect as an inhibitor / antagonist is observed at least with respect to TK 3, 4, 5, 6, 9. A decrease in TK activity is observed when TK are expressed under the influence of many TK ligands ίη igo and ίη ugo. It has also been found that they are useful in treating conditions induced by TB ligands, such as, for example, lipopolysaccharides (cytokines and hyperthermia). It was also found that they are useful in the treatment of diseases in which there is an increased expression of various jupe-like receptors, for example, with sepsis, multiple myeloma, malaria, multiple sclerosis, retrobulbar neuritis, chronic obstructive pulmonary disease, etc.

The invention is illustrated by the following examples without limiting the scope of the invention.

I. In accordance with the invention, the following examples describe the composition of the pharmaceutical composition, the manufacturing method, its IRIS characteristic, safety and tolerability, methods of use and outcome of treatment. The following are illustrative examples of the present invention, and the scope of the present invention should not be limited to them.

Example 1. Pharmaceutical compositions.

A. Each dose of 0.1 ml of therapeutic agent contains Mycobacterium No. 0.50x10 ’(heat inactivated)

Sodium chloride I. R. Tween 80

0.90% w / v 0.1% w / v

Thiomersal I. R.

Water for injection I. R.

B. Each dose of 0.1 ml of therapeutic agent contains

0.01% w / v (as preservative) as needed up to 0.1 ml

Mycobacterium νν (heat inactivated) Sodium chloride I. R.

Triton x 100

Thiomersal I. R.

Water for injection I. R.

0.50 x 10 ⁹

0.90% w / v

0.1% wt.

0.01% w / v (as preservative) as needed up to 0.1 ml

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C. Each dose of 0.1 ml of therapeutic agent contains

Mycobacterium νν 0.50 χ 10 ⁹ (heat inactivated)

Sodium chloride I. R. 0.90% wt./about.

Thiomersal I. R. 0.01% wt./about. (as a preservative)

Water for injection I. R. as needed up to 0.1 ml

Ό. Each dose of 0.1 ml of therapeutic agent contains

Mycobacterium νν extract after sonication from 1 x ¹⁰ Mycobacteria ul /

Sodium chloride I. R. 0.90% wt./about.

Thiomersal I. R. 0.01% wt./about. (as a preservative)

Water for injection I. R. as needed up to 0.1 ml

E. Each dose of 0.1 ml of therapeutic agent contains a methanol extract of 1x10 '° mycobacteria νν Sodium chloride I. R. 0.90% wt./about.

Thiomersal I. R. 0.01% wt./about. (as a preservative)

Water for injection I P as needed up to 0.1 ml

R. Each dose of 0.1 ml of therapeutic agent contains Chloroform extract 1x1O ¹⁰ mycobacteria \ ν Sodium chloride I. R. 0.90% wt./about.

Thiomersal I. R. 0.01% wt./about. (as a preservative)

Water for injection I. R. as needed up to 0.1 ml

C. Each dose of 0.1 ml of therapeutic agent contains Acetone extract 1x10 ^{1 <,} Mycobacteria νν Sodium chloride I. R. 0.90% wt./about.

Thiomersal I. R. 0.01% wt./about. (as a preservative)

Water for injection I. R. as needed up to 0.1 ml

H. Each dose of 0.1 ml of therapeutic agent contains

Ethanol extract 1x10 ¹ “mycobacteria and /

Sodium chloride I. R. 0.90% wt./about.

Thiomersal I. R. 0.01% wt./about. (as a preservative)

Water for injection I. R. as needed up to 0.1 ml

I. Each dose of 0.1 ml of therapeutic agent contains

Lytic treated extract 1x10 “mycobacteria νν Sodium chloride I. R. 0.90% wt./about.

Thiomersal I. R. 0.01% w / v. (as a preservative)

Water for injection I. R. as needed up to 0.1 ml

1. Each dose of 0.1 ml of therapeutic agent contains

Mycobacterium νν 0.5 x 10 ^t (heat inactivated)

Mycobacterium νν extract obtained by destruction, solvent extraction or enzymatic extraction, 1x10 ⁵ mycobacteria νν Sodium chloride I. P. 0.90% wt.

Thiomersal I. R. 0.01% wt./about. (as a preservative)

Water for injection I. P as much as necessary up to 0.1 ml

I. R. = International Pharmacopoeia

Example 2. A method of manufacturing a pharmaceutical composition.

A. Cultivation of mycobacteria \ ν.

1) Preparation of culture medium.

Mycobacterium ν is cultivated on a solid medium similar to the L-medium (Lowenstein-Jensen medium) or a liquid medium similar to Middlebrook medium or Sathon liquid medium.

Middlebrook is enriched to improve yield. Preferably, it is enriched by the addition of glucose, bactotryptone and Β8Ά (bovine serum albumin). They are used in a ratio of 20: 30: 2 preferably. An enrichment medium is added to Middlebrook's medium. This is preferably carried out in a ratio of 15: 1 to 25: 1, more preferably in a ratio of 20: 1.

2) Work with a bioreactor.

a) Preparation of the vessel.

The internal contacting parts of the vessel (joints, mechanical seals, o-rings / grooves for gaskets, etc.) must be properly rinsed to prevent any contamination. Fill the vessel with 0.1 n. ΝαΟΗ and leave in this state for 24 hours to remove pyrogenic substances and other impurities. Then the vessel is washed first with acidified water, then with ordinary water. Finally, the vessel is rinsed with distilled water (3 times) before preparing the medium.

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b) Sterilization of the bioreactor.

A bioreactor containing 9 l of distilled water is sterilized with hot steam (not directly). Similarly, the bioreactor is sterilized again with Middlebrook medium. Other additional bottles, inlet / outlet air filters, etc. autoclave (twice) at 121 ° C for 15 minutes Before use, all this is dried at 50 ° C in an oven.

c) Environmental parameters.

1. Temperature: 37 ± 0.5 ° C

2. pH 6.7-6.8 at the beginning.

B. Collection and concentration.

This is usually done by the end of 6 days after cultivation under aseptic conditions. Concentration of cells (collection of sediment) is performed by centrifugation.

C. Cell washing.

The precipitate thus obtained is washed at least three times with standard saline. It can be washed with any other liquid, preferably isotonic.

Ό. Addition of a pharmaceutically acceptable carrier.

Pyrogen-free standard saline is added to the precipitate. As a pharmaceutical carrier, any other pyrogen-free isotonic liquid may be used. The carrier is added in such an amount as to obtain the desired concentration of active principle in the final form.

E. Addition of preservative.

To keep the product free of other contaminating bacteria during its shelf life, a preservative is added. A preferred preservative is thiomersal, which is used at a final concentration of 0.01% w / v.

R. Final sterilization.

Final sterilization can be carried out by various physical methods, such as using heat, or ionizing radiation, or sterile filtration.

The heat may be in the form of dry heat or wet heat. In addition, it may be in the form of boiling or pasteurization. The ionizing radiation may be ultraviolet or gamma rays or microwave radiation or any other form of ionizing radiation. It is preferable to autoclave the final product. This can be done before / after filling the final package.

C. Quality control.

1. The substance is evaluated for cleanliness, sterility.

2. Microorganisms are monitored for acid resistance after Gram staining.

3. Inactivation test: it is carried out by culturing the product on an S-medium to detect any living microorganism.

4. Pathogenicity and / or contamination of the pathogen. Cultured microorganisms infect Ba1b / s mice.

None of the mice should die, everyone should stay healthy and give weight gain. No macroscopic or microscopic lesions should be observed in the liver, lung, spleen or any other organs after the animals are killed, up to 8 weeks after treatment.

5. Biochemical test.

The microorganism is subjected to the following biochemical tests:

a) urease;

b) hydrolysis of tween 80;

c) niacin test;

b) nitrate reduction test.

The microorganism gives negative results in the urease test, test for hydrolysis of tween 80 and niacin test. Positive according to nitrate reduction test.

H. Preparation of components of mycobacteria \ ν.

The components of mycobacteria ν can be prepared for the task of the present invention by:

I. destruction of cells;

2. solvent extraction;

3. enzymatic extraction.

Cell destruction can be accomplished by sonication or by using a high-pressure fractionation device (Ggasbopots1sg) or by using an ingredient to create osmotic pressure (oktobs rtekkite 1pdgsb1sp1).

Solvent extraction can be carried out using any organic solvent such as chloroform, ethanol, methanol, acetone, phenol, isopropyl alcohol, acetic acid, urea, hexane, etc.

Enzymatic extraction can be carried out using enzymes that can

- 5 023046 to destroy cell walls / membranes. They are usually proteolytic in nature. Preferred enzymes are lyticase and pronase enzymes. For the purpose of the invention, instead of the microorganisms of mycobacteria, they can use only the cellular components of mycobacteria, or they can be added to the product containing mycobacteria.

The addition of cellular components increases the effectiveness of the product.

In all examples, the mycobacterium te described in Example 1c is used, which is heat inactivated mycobacterium te in an amount of 0.50 x ^{10 9} in 0.1 ml.

II. Examples showing a reduction in TEK induction by using the pharmaceutical compositions of the present invention.

Example 1

TK stimulation is tested by evaluating the activation of ΝΡ-κβ (nuclear factor κβ) in HEK293 cells expressing a given TK. The antagonistic activity of a pharmaceutical composition containing 0.5 x ^{10 9} cells of heat-inactivated mycobacterium te in 0.1 ml of isotonic solution is tested against human TK (TK): 2, 3, 4, 5, 7, 8, and 9.

The following TLC ligands are used in this study:

YTRK2: NK1.M (inactivated by heating 1,1-1epa toposudedepa) at a concentration of 2x10 ⁷ , 1 x 10 ⁷ , 2x10 ⁶ and 1 x 10 ⁶ cells / ml;

LTK.3: poly- (RS) (polyinosine: polycytidylic acid) at a concentration of 20 and 10 ng / ml;

Lt4: L3 E. coli K12 at a concentration of 2 and 1 ng / ml;

IT1.K5: flagellin Z. 1urYShigshsh at a concentration of 20 and 10 ng / ml;

LTK7: loxoribine at a concentration of 1 and 0.5 mm;

IT1, K8: 88-40 (single-stranded RNA40) at a concentration of 5 and 3 μg / ml;

RK9: CfO ΟΏΝ 2006 at a concentration of 50 and 20 ng / ml.

General technique.

The reporter gene for secreted alkaline phosphatase is under the control of a promoter induced by the transcription factor ΝΡ-κΒ. Stimulation of T1.K in this screening is tested by evaluating the activation of ΝΡ-κΒ in HEK293 cells expressing a given T1, K. This reporter gene allows monitoring of signal transduction via TK based on ΝΡ-κΒ activation. In wells of a 96-well plate (total 200 μl) containing the corresponding number of HEK293 cells (25000-50000 cells / well), 20 μl of heat-inactivated mycobacteria, as well as TBK ligands, are added. The media added to the wells are designed to detect the ΝΡ-κΒ-induced expression of ZEAP (secreted alkaline phosphatase). After 16-20 hours of incubation, in order to detect the induced ΝΡ-κΒ-activity, ΟΙ2 (optical density) is measured at 650 nm on a Vesktap Soikeg AE 340C Alabhapse E1ec1og.

The results are presented in tabular form, as mentioned below. Column (A) shows the activity induced by TB ligands. Column (B) demonstrates the activity induced by the TB ligand in the presence of a composition containing mycobacteria te (Mte). The last column shows the antagonism expressed in%, due to the action of mycobacterium te in comparison with only one TLC ligand.

Tk POSITIVE CONTROL (ligand T1_P) (A) POSITIVE CONTROL * Mu / (B) B / A,% Antagonism, % tyrs 1,881 1,445 76.8% 23.2% THP4 1,207 0.602 49.87% 50.13% T1_P5 2,227 1,134 50.92% 49.08% T1_P7 1,503 1,141 74.57% 25.43% ΤΙ.Ρ8 0.591 0.431 72.92% 27.08% ΤΙ.Ρ9 1,979 0.135 0.07% 99.93% ΤΙ.Ρ2 2.21 2.17 98.19% 1.81%

These data indicate TK antagonism upon stimulation of K 3, 4, 5, 7, 8, 9 with the TK ligand. In this experiment, antagonism against T1, K2 is not observed.

Example 2

Mice 8-10 weeks old were euthanized and splenocytes were isolated from the spleen. Spleen cells were cultured with LBP and a combination of LBP with heat inactivated mycobacterium cells in different concentrations. Cells were cultured in 1640 media from KPM1 (Coate11 Ragk Metopa1pay1i1e).

After 48 hours, cells were harvested and monitored for the expression of different TBTs. The expression of TLC is monitored by amplification of a specific mRNA from a cell lysate (Ce11σΟΝΑ II kit, AbLup) using TK-specific primers (K & aa! Eta).

Amplified products were monitored on a 1.5% agarose gel using ethidium bromide staining.

It was found that the expression of TK 3, 4, 5, 6, and 9 is reduced if the cells are exposed to the mycobacteria te + L3P as compared with only L3P. No effect was found only for T1.K1. (Fig. 1). So

- 6 023046 thus, a pharmaceutical composition containing mycobacterium ν exhibits antagonistic activity with respect to Lp-induced induction of TK 3, 4, 5, 6 and 9. For TK1 induced by L8, no effect was found.

Example 3

The administration of the poly-TLC ligand to mice induces the expression of TLC 1, 3, 5, 6, 9 in splenocytes collected and analyzed after 7 days.

Splenocytes expressing TBK 1, 3, 5, 6, 9, after stimulating them with ίη νίίτο for 48 h using 0.5x10 ⁵ or more heat-inactivated mycobacteria \ ν show the absence of TBK expression 3, 5 and 6 (100% decrease in expression of TB 3, 5, and 6). The results are shown in FIG. 2.

Thus, the above examples demonstrate a decrease in the induction of TB through the action of the pharmaceutical composition of the present invention.

III. Examples demonstrating antagonistic activity against the action of TBK ligands.

Example 1. The effect on Lp-induced cytokines.

Mice aged 8-10 weeks were killed and splenocytes were isolated from the spleen. Spleen cells were cultured with L8 and a combination of L3 with heat inactivated Mycobacterium v cells in various concentrations. Cells were cultured in 1640 media from KPM1.

The supernatant was analyzed for cytokines like Tyr-alpha (tumor necrosis factor-alpha) and и-gamma (interferon-gamma).

In studies of ίη νίίτο, mycobacterium ν, when used together with lipopolysaccharide (L8), reduces the induction of TYP alpha under the influence of L2 and also reduces the secretion of гам-gamma. The level of observed inhibition is significant and so great that secretion decreases to the basal level (complete inhibition).

Example 2. The effect on Lp-induced hyperthermia.

Rabbits were prepared in the same way as for the pyrogenicity test, and temperature was monitored.

Rabbits were given an intravenous injection of E. soy lysate to mimic endotoxin / Lp-induced hyperthermia. Two hours later, they were divided into a control group and a treatment group. The treated group received an injection of 2 ml of a pharmaceutical composition containing heat inactivated mycobacterium ν (0.5 x ^{10 9} cells in 0.1 ml). The received data is reproduced three times. Animals in the treated group showed a decrease in temperature, while the control animals remained at an elevated temperature. The effect, stably maintained until the end of the experiment, is shown in FIG. 3.

Thus, the above examples demonstrate the antagonistic effect of the pharmaceutical composition of the present invention with respect to the action of TLC ligands.

IV. Examples showing usefulness in diseases in which increased expression of TB occurs.

(BUT). Improved survival in E. soy-induced sepsis in mice.

Parenteral injection of live E. soy to mice causes sepsis with a mortality rate of 100%. The pharmaceutical composition of the present invention, containing 0.5 x ^{10 9} cells of heat-inactivated mycobacterium ν, was evaluated (Μν) for its effectiveness in this mouse model.

Animals were injected with E. soybean suspension, then mycobacterium ν in / in (intravenous) or / and (intradermal) methods in various combinations with or without dexamethasone and amoxicillin. The greatest improvement in survival is observed in animals treated with mycobacterium ν with glucocorticoid and amoxicillin.

Experiment 1 (intraperitoneal administration).

Mice were injected with 1 ml of live E. soy (20 ΘΌ) intraperitoneally. Mice were treated using a different combination of amoxicillin (500 mg) and dexamethasone (2.0 mg) therapies (Fig. 4). There were 10 animals in each group. In the control group, all animals died within 48 hours (group I). Interventions showed improved survival (Π-νΠ group). 100% survival was observed when dexamethasone + amoxicillin was combined with mycobacterium ν (0.1 ml) delivered intradermally (group IV). It was followed by a group treated with the same drugs, but with mycobacterium ν intravenously administered (group V). The results are presented graphically in FIG. 4.

Experiment 2 (intraperitoneal administration).

In the second experiment, the dose of amoxicillin was reduced from 500 mg / kg to 70 mg / kg (Fig. 5). Again, the best results were observed for group II with mycobacterium ν, administered intradermally along with steroids and antibiotics, followed by group III with an intravenous route of administration. The results are presented graphically in FIG. 5.

A comparison of experiments 1 and 2 shows that the amount of antibiotic is significant. This is important from the point of view of the clinic, since in the treatment of sepsis, a large dose of the antibiotic is used, and not the usual dose used to treat other infections.

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Experiment 3 (intravenous administration).

In a third experiment, sepsis was induced by the intravenous route and dexamethasone levels were reduced from 2 to 0.5 mg / kg. The data obtained confirm that the combination of steroids + antibiotics + mycobacteria \ ν (group VI) provides improved survival. These three experiments showed that the use of mycobacteria ν improves survival in sepsis. The results are presented graphically in FIG. 6.

(B) Model malaria R. Jetdejk

It is known that dysregulation of the immune system in malaria P. £ a1c1ratit induces sepsis-like syndromes. In animals infected with a sudden and rapidly developing form of malaria caused by a strain of P. brue! ΑΝΚΑ, observe an identical situation. Previous work done at the Indian Institute of Sciences (No. c) in Bangalore showed that the combination of mycobacteria ν + artiter results in 70% survival compared to 0% in the control group receiving only artiter or only placebo. Thus, mycobacterium ν is able to reverse the sepsis-like syndromes caused by malaria infections.

Thus, the above examples (ΐν-Α and ΐν-Β) demonstrate the usefulness of the pharmaceutical composition of the present invention in diseases in animals in which there is an increased expression of TBA.

(C). Sepsis in humans.

It has been established that a product that promotes excretion (1eab rgobis!) Is useful in the treatment of a chronic infectious disease. It was also found that it is useful in the resorption of steroid-resistant granulomas, pleural effusion, hydropneumothorax, retrobulbar neuritis, etc.

Its effect is also appreciated in the treatment of sepsis.

Example 1

In an elderly patient, a 65-year-old man with resistant myeloma, sepsis was detected and the patient was connected to a ventilator for 6 weeks. For treatment, he received a higher dose of antibiotics (penems), EPO (erythropoietin), OM C8P (granular macrophage colony stimulating factor), and daily platelet transfusion. Despite this, he experienced a sharp deterioration in the course of the disease with a progressive decrease in the number of SI4. He was injected with 0.2 ml of mycobacterium ν intradermally in two divided doses above the deltoid muscle. After 48 hours, he was disconnected from the ventilator. He did not need any platelet infusion.

Example 2

A weakened young man with chronic tuberculosis (TB) of the lungs develops a bacterial infection of the lung that leads to sepsis. He did not respond to traditional therapy. He was injected with mycobacterium ν (5 ml, iv) for 5 days.

This led to a cure for sepsis and infection and discharge from the hospital. His condition remained stable without relapse for the next 6 weeks. Both of these examples confirm that mycobacterium ν is useful in the treatment of sepsis in humans.

(AND). Retrobulbar neuritis.

In a 56-year-old male patient with retrobulbar neuritis, residual visual impairment (te81bia1 U18ia1 bEPi) was detected after treatment with methylprednisolone, 1 g, administered intravenously daily for 3 days. Vision remained stable at 6/36 and 6/18, respectively, in the right and left eye, respectively. He was injected with 5 ml of mycobacteria ν (prepared according to the invention) in 500 ml of an isotonic solution intravenously, again by infusion. Vision improved, reaching 6/12 and 6/9, respectively, in the right and left eye 10 days after the start of treatment.

(E). Multiple sclerosis.

A 28-year-old female patient had paralysis of both lower extremities with a level of motor ability of 0 (ro \\ ex dhabe 0) due to multiple sclerosis. He did not respond to treatment for 6 months. During this period of time, she did not receive any other treatment.

She was injected with 2 ml of mycobacterium ν daily in 100 ml of an isotonic solution intravenously for three days. When viewed after 15 days, she showed signs of recovery with a level of motor ability II for both lower limbs.

(R). Chronic obstructive pulmonary disease.

The use of a pharmaceutical composition containing mycobacterium ν in the treatment of chronic obstructive pulmonary disease leads to a decrease in excretion, a decrease in the degree of infection, and a decrease in the number of complications requiring hospitalization, according to examples demonstrating its effect on ΡΕν1 and PEPC.

Experiment 1.

Patients with chronic obstructive pulmonary disease were injected with 0.1 ml of mycobacterium ν intradermally every two weeks for 2 months. This led to an improvement in the one-second volume of air during forced expiration (ΡΕν1) by more than 50% in 5 out of 9 patients, in the rest the improvement was more than 25%. An improvement in maximum expiratory flow (PERC) also followed

- 8 023046 of this pattern.

Experiment 2.

patients with chronic obstructive pulmonary disease were injected intramuscularly with 0.5 x ^{10 9} cells of heat-inactivated mycobacteria after they were suspended in 1 ml of isotonic solution.

As a result of a single injection, improvement in REU1 was observed in 13 of 16 patients when tested on the 15th day after injection (more than 50% in 7, more than 25% in 6). This improvement reached its peak after 4-6 weeks. Similarly, improvement in PEPC was observed in 11 of 16 patients.

Experiment 3.

patients with chronic obstructive pulmonary disease were injected with 0.5 x ^{10 9} heat inactivated mycobacteria cells into the nasal mucosa by means of a nasal spray (0.1 ml). An improvement in REU1 of more than 25% was observed in 5 out of 6 patients, while 3 patients showed an improvement of more than 50%.

An improvement in PERC of more than 25% was observed in 4 of 6 patients, while 2 of them showed an improvement of more than 50%.

Experiment 4.

Ten patients with chronic obstructive pulmonary disease were once injected with 0.5 x ^{10 9} heat-inactivated mycobacteria cells, suspended in 3 ml of diluent, into the lung by spraying. Improvement in REU1 (by more than 25%) was observed in all 10 patients.

The effect is observed for more than 6 weeks. The improvement in PERC was more than 25% in 8 patients, while 4 of them achieved an improvement of more than 50%.

(FROM). Multiple myeloma

The patient - a woman of 70 years with a myeloma that is immune to traditional methods of treatment, the disease progressed. She experienced severe bone pain. The pain was so intense that she could not walk. Mycobacterium te (0.1 ml mycobacterium te) was administered intradermally every month. A remission of the disease was achieved. After three months of therapy, she did not show symptoms of the disease, and she could move freely. Her hemoglobin index improved from 5.5 to 7.5 g-%.

Thus, the above examples (from RU-C to 1U-O) demonstrate the beneficial effects of the pharmaceutical composition of the present invention in the treatment of diseases in humans in which there is an increased expression of TEK.

Examples ΙΙ-1 - ΙΙΙ-3 illustrate the poly-TEK antagonistic activity of mycobacteria or its components upon induction by known TK agonists, synthetic, such as CpC, ΘΌΝ, or natural, such as lipopolysaccharide. Examples ΙΙ-1 and ΙΙ-2 also illustrate the antagonistic activity of mycobacterium te and its components with respect to the action of TK ligands like lipopolysaccharide, E-CoH, etc. Examples ДУ-А, Ιν-Β also illustrate the usefulness of mycobacteria of one or its components in the treatment of diseases in which there is an increased expression of TEK. Examples of RU-S, RU-E also illustrate the positive effects of mycobacteria on one or its components in the treatment of a disease like sepsis, retrobulbar neuritis, multiple sclerosis, chronic obstructive pulmonary disease, multiple myeloma.

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Claims (15)

CLAIM 1. The use of heat inactivated MuObac1epis (mycobacteria te) to reduce the induced TBR 3, 4, 5, 6, 7, 8, 9 activity. 2. The use according to claim 1, where TBR activity is induced in diseases. 3. The use according to claim 1, where the TBR activity is induced by ligands of the TBR. 4. The use according to claim 3, where the TBR ligand is a ligand of natural origin, for example a lipopolysaccharide or endotoxin, or synthetic, for example CPO, ΟΌΝ (oligodeoxynucleotide). 5. A method of reducing induced TBA activity in diseases using mycobacteria, those comprising administering a therapeutic amount of a pharmaceutical composition comprising heat inactivated mycobacteria. 6. The use according to claim 1, where TBR activity is induced in sepsis. 7. The use according to claim 1, where TBR activity is induced in chronic obstructive pulmonary disease. 8. The use according to claim 1, where TBR activity is induced in multiple myeloma. 9. The use according to claim 1, where TBR activity is induced in multiple sclerosis. 10. The use according to claim 1, where TBR activity is induced in retrobulbar neuritis. 11. Use of a therapeutically effective amount of heat inactivated mycobacteria for the treatment of sepsis. 12. Use of a therapeutically effective amount of heat inactivated mycobacteria for treating chronic obstructive pulmonary disease. 13. Use of a therapeutically effective amount of heat inactivated mycobacteria for the treatment of multiple myeloma. 14. Use of a therapeutically effective amount of heat inactivated mycobacteria for the treatment of multiple sclerosis. 15. The use of a therapeutically effective amount of heat inactivated mycobacteria for the treatment of retrobulbar neuritis. Tk Ι_Ρ5 + 10® cells Μνν FIG. one FIG. 2 Pyrogenicity test FIG. 3 - 12 023046 FIG. 4 I. E. CoH, II. E. coH + dexamethasone (0.5 ml 4 mg / ml), III. E. CoH + amoxicillin (500 mg / kg), IV. E. coH + dexamethasone + amoxicillin + mycobacteria \ ν 0.1 ml i / d, V. E. coH + dexamethasone + amoxicillin + mycobacteria ν 0.1 ml iv, VI. E. coH + dexamethasone + mycobacteria ν 0.1 ml and / d, VII. E. coH + dexamethasone + mycobacterium ν 0.1 ml iv Mortality FIG. 5 I. E. CoH, II. E. coH + dexamethasone (2.0 mg) + amoxicillin (70 g / kg) + mycobacterium ν 0.1 ml i / d, III. E. coH + dexamethasone (2.0 mg) + amoxicillin (70 mg / kg) + mycobacterium ν 0.1 ml iv, IV. E. coH + amoxicillin (70 mg / kg) + mycobacterium ν 0.1 ml i / d, V. E. coH + amoxicillin (70 mg / kg) + mycobacterium ν 0.1 ml iv I. E. CoH, II. E. coH + dexamethasone (0.5 mg / kg), III. E. coH + dexamethasone (0.5 mg / kg) + mycobacterium ν 0.1 ml i / d, IV. E. coH + amoxicillin (70 mg / kg), ^ E. coH + dexamethasone (0.5 mg / kg) + amoxicillin (70 mg / kg), VI. E. coH + dexamethasone (0.5 mg / kg) amoxicillin (70 g / kg) + mycobacterium 0.1 ml and / d